Gastrointestinal injury usually starts in the superficial mucosa. We investigated whether leukocyte-endothelial interactions were greater in the gastro-intestinal mucosa than the submucosa and muscularis in control tissue and following upregulation of adhesion molecules with endotoxin and after chemical insult with NSAID. Inactin-anesthetized rats were given either endotoxin or flurbiprofen or NO-flurbiprofen after which ICAM-1 and P-selectin expression was measured with the dual label antibody technique. Leukocyte-endothelial interactions in the different gastric layers were assessed after endotoxin using intravital microscopy. Endotoxin caused a 2-3 fold increase in ICAM-1 expression in the stomach and duodenum. There was however a gradient in expression across the gut wall with the level of expression in the superficial mucosa (per g) being only 10-25% of that in the deeper layers, in both control and endotoxin treated animals. Constituitive expression of P-selectin in control animals was barely detectable. Endotoxin caused a modest increase in the mucosal P-selectin but a very significant increase in the deeper layers. Flurbiprofen caused a slight upregulation of ICAM-1 in the gastric mucosa and duodenum while NO-flurbiprofen had no affect on expression. Intravital microscopy revealed no adhesion and virtually no leukocyte rolling in the vessels of the gastric mucosa in spite of endotoxin treatment. There was however some adhesion and significant leukocyte rolling in the submucosa and muscularis. Thus, the superficial gastric and duodenal mucosal microcirculations have a much lower density of ICAM-1 and P-selectin and less leukocyte-endothelial interactions than occurs in the deeper layers of the gut wall even during stimulated upregulation with endotoxin.